The present invention relates to the construction of adenovirus vectors that have increased safety and stability for gene transfer in mammalian cells. The vector system described herein is an improvement and modification of the helper-dependent system, described in copending patent application Ser. No. 08/473,168.
Adenoviruses (Ads) are a family of DNA viruses characterized by icosahedral, non-enveloped capsids containing a linear DNA genome.
The human adenovirus type 5 (Ad5) has a linear, double-stranded genome of approximately 36 kb, divided into early and late viral functions (see Berkner 1992, Curr. Topics Micro. Immunol. 158:39-66). A representative Adenovirus 5 (xe2x80x9cAd5xe2x80x9d) genome for use with the embodiments of the present invention is a 36 kB linear duplex. Its sequence has been published. (Chroboczek, J., Bieber, F., and Jacrot, B. (1992) The Sequence of the Genome of Adenovirus Type 5 and Its Comparison with the Genome of Adenovirus Type 2, Virology 186, 280-285; hereby incorporated by reference).
Upon infection of permissive cells, the first region transcribed from the Ad5 viral genome, E1A at the left end of the conventional map, encodes proteins that are involved in transactivation of other viral early and late genes. E1B, also at the left end of the genome, encodes proteins that regulate host cell and viral RNA and protein synthesis, and protect cells from E1A-induced apoptosis. Thus, E1 functions encoded by E1A and E1B are essential for viral replication. E1-deleted virus can be propagated in the 293 cell line which contains and expresses E1 of Ad5 (Graham et al. 1977).
Removal of the essential early regions 1A and 1B (E1A and E1B) of Ad5 generates conditional helper-independent Ads that can be grown and propagated in the E1-complementing 293 cell line (Graham et al. 1977, J. Gen. Virol. 36:59-72). Foreign genes have been cloned into the replication-defective Ads, and these vectors have been used extensively for the delivery of genes into mammalian cells for gene therapy, as recombinant viral vaccines, or for general purpose expression vectors for experimental studies. Ads also have the advantage that they are well characterized both genetically and biochemically, easy to manipulate, and can be grown to a very high titer. Furthermore, adenovirus is a relatively safe vector that has not been associated with any neoplastic disease, and usually causes relatively mild infections in immuno-competent individuals.
E1-deleted Ad vectors can accommodate DNA inserts of xcx9c4.7 kb (up to 105% of the wild-type genome), and deletions in the non-essential E3 region can further increase the cloning capacity to xcx9c8 kb (Bett et al. 1993, J. Virol. 67:5911-5921). However, Ad vectors with DNA inserts that increase the genome size to greater than 105% of wild-type DNA content are either non-viable or unstable, and frequently undergo DNA rearrangements to reduce the overall size of the vector (Ghosh-Choudhury et al. 1987, EMBO J. 6:1733-1739; Bett et al. 1993, J. Virol. 67:5911-5921). This is presumably due to a destabilization of the capsid because of the increased DNA content. Thus, the size of DNA inserts in xe2x80x9cfirst generationxe2x80x9d Ad vectors (i.e., E1-deleted and/or E3 deleted) is limited by the necessity to retain sufficient Ad coding sequences to allow helper-independent growth, limiting the size of xe2x80x9cnon-essentialxe2x80x9d regions that can be deleted from the genome, and the need to maintain virion stability.
Stability of the adenovirus capsid is conferred, at least in part, by protein IX (pIX). pIX has been shown to be associated with the hexons that make up the xe2x80x9cfacetsxe2x80x9d of the icosahedron (Furcinitti et al. 1989, EMBO J. 8:3563-3570) Although originally thought to be dispensable for virion formation (Colby and Shenk 1981, J. Virol. 39:977-980), pIX is required for the packaging of full-length viral DNA molecules (Haj-Ahmad and Graham 1986, J. Virol. 57:267-274). Deletion or inactivation of pIX results in virions that are heat labile with capsids that can accommodate only 35 kb of viral DNA (xcx9c97% of the wild-type genome). Thus, deletion or inactivation of pIX provides a means of selecting for virions that contain viral DNA that is less than the size of the wild-type genome.
Previously, the lower limit of adenovirus DNA necessary to achieve Ad DNA packaging could not be identified due to the necessity for retaining sufficient protein-coding regions to enable the production of all of the proteins required for Ad DNA replication and virion formation. The development of helper-dependent systems has alleviated this problem. In the helper-dependent systems, a helper virus provides all of the functions necessary in trans for the packaging of an helper-dependent vector, which lacks virtually all virus specific coding sequences. The helper-dependent vector contains only those cis-acting elements required for viral DNA replication and packaging. Since the sequences required for Ad DNA replication and packaging are contained within xcx9c500 bp of the left and right ends of the genome (Grable and Hearing 1992, J. Virol. 66:723-731), helper-dependent vectors can, in theory, range in size from a few hundred base pairs to greater than the size of wild-type Ad, potentially carrying up to xcx9c37 kb of foreign DNA. However, it has been demonstrated that Ad vectors that have substantially less DNA than wild-type Ads undergo DNA rearrangements and multimerization (Fisher et al. 1996, Virol. 217:11-22).
Despite all of the advantages of first generation Ad vectors as vectors for the delivery of foreign genes into mammalian cells, current helper-independent vectors retain many viral genes that, when expressed at low levels, may contribute to the induction in the host of an immune response against the transduced cell (Dong et al. 1996, Hum. Gene Ther. 7:319-33 1), resulting in the elimination of the transduced cell. The immune response will ultimately limit the usefulness of current vectors for the treatment of genetic diseases, such as cystic fibrosis, due to the requirement for long term, stable expression in order to correct the genetic deficiencies. Attempts to reduce the expression of viral genes, by the elimination of most, if not all, viral-specific coding sequences, have led to the development of the helper-dependent systems for the generation of Ad vectors (Mitani et al. 1995, Proc. Natl. Acad. Sci. 92:3854-3858; Fisher et al. 1996, Virol. 217:11-22; Kochanek et al. 1996, PNAS 92:5731-5736; Parks et al. 1996, Proc. Natl. Acad. Sci. in press). Previously, we developed a helper-dependent system that utilized a helper virus that had a packaging signal flanked by loxP sites (Parks et al. 1996, Proc. Natl. Acad. Sci. in press). The general principle is outlined in FIG. 1. Upon infection of a 293 cell line that constitutively expressed the Cre recombinase (293Cre; Chen and Graham, unpublished results), the packaging signal was efficiently excised from the helper virus rendering it unpackageable. However, the helper virus DNA was able to replicate and provide all of the functions necessary in trans for the packaging of a helper-dependent vector, which contained only those cis-acting elements required for viral DNA replication and packaging. Serial passage of the helper-dependent vector in helper-virus infected 293Cre cells allowed us to produce large quantities of the helper-dependent vector (1010 transducing particles from 4xc3x97108 293Cre cells with an initial level of contamination with helper virus of approximately 0.3-1%). After fractionation on CsCl buoyant density gradients, final vector preparations contained less than 0.01% helper virus contamination, a level that is lower than in all other helper-dependent systems reported to date. The contamination of vector with helper virus that is observed is caused by helper virus DNA (xcx9c10%) that escapes the Cre-mediated excision event, and can therefore be packaged into infectious virions. At present, it is not known why these DNAs are not cleaved by Cre, but it may be due to saturation of the Cre protein in the 293Cre cells. Regardless of the reason for the helper virus contamination of vector stocks, it is apparent that modifications to the system are desired to eliminate the remaining helper virus. It is an object of the present invention to provide an improved method for preparing helper-dependent vectors. The invention herein may be used independently for vector growth or may be combined with the Cre/loxP helper-dependent system to provide a means for vector production without contaminating helper virus.
It is an object of this invention to provide a simple and useful improved helper-dependent adenovirus vector system by which high capacity adenovirus cloning vectors may be developed. The invention makes use of the DNA size packaging constraints imposed on a pIX-defective Ad virion that prevent such virions from packaging DNA larger than approximately 35 kb. This constraint can be used to develop helper adenoviruses that do not package their DNA. Additionally, one embodiment of the invention combines this methodology with the Cre-loxP helper-dependent system to decrease the quantity of helper virus in vector preparations. Such helper virus, though not able to package DNA into infectious virions, can replicate and provide all of the functions in trans for the packaging of a second vector known as the helper-dependent adenovirus vector that lacks substantial portions of the Ad coding sequences.
In accordance with the present invention, helper viruses having genomes of a size greater than the upper limit for packaging in a pIX-defective virion are provided. One embodiment of the present invention is the construction of a helper virus from two vectors. Preferably, the first vector includes a circularized, modified human adenovirus type 5 (Ad5) genome that is deleted for, or contains mutations in, the DNA sequence encoding pIX. This first vector is combined with a second vector containing overlapping viral DNA sequences to generate infectious Ad5, known as a helper virus having a modified pIX, and a genome size greater than the upper limit for packaging in a pIX-defective virion. Alternatively, the size of the helper virus can be increased by the insertion of additional DNA sequences into the adenoviral genome, known as xe2x80x9cstufferxe2x80x9d DNA. Bacterial plasmids are preferred vectors for obtaining the helper virus. However, other vectors may be employed to construct the helper virus, such as, for example, yeast plasmids.
Although not able to produce adequate proteins, particularly pIX, to permit its own packaging, the helper virus described herein, is able to produce all of the functions required for the packaging of a helper-dependent viral vector having a genome of appropriately reduced size (i.e., less than about 35 kb) and lacking substantial portions of the viral genome so that the helper-dependent vector DNA can be packaged in pIX-defective virions. Such helper virus and helper-dependent vector DNA may replicate when coinfected into appropriate host cells, but only the helper-dependent vector DNA can be packaged. Optionally, certain regions of the vectors and resulting viruses may be deleted, such as sequences in the Ad E1 or E3 regions that can be omitted from the viral genome without preventing the viral genome from replicating in such cells as may be permissive for replication of said genome in the form of infectious virus.
The vectors used to generate the helper virus and the resulting helper viruses may also contain sequences that can be recognized by a site specific recombinase. For example, Cre recombinase is suitable for use with the present invention. Recombination catalyzed by Cre acting on an appropriately constructed viral genome will result in the excision of a nucleotide sequence, known as the packaging signal ("psgr"), near the left end of the viral genome, that is required for the packaging of adenoviral DNA into infectious particles. Use of the Cre recombinase in this and other examples is not meant to be limiting as other site specific recombination systems do exist and might also be employed. An example, not meant to be limiting, is the use of the yeast FLP recombinase and its recognition sequences (O""Gorman et al. 1991, Science 251:13-51), which could readily be substituted for the Cre protein in this and other examples.
Alternatively, vectors for generating the helper virus and the resulting helper virus having mutations in the Ad packaging signal that result in reduced efficiency of DNA packaging are suitable for use with the present invention.
A second embodiment of the present invention provides helper-dependent vectors having Ad genomes that are unable to replicate as viruses in the absence of viral products provided by a second virus, i.e., the helper virus. In one embodiment of the present invention helper-dependent vectors are derived from bacterial plasmids that contain only those viral sequences required for the replication and packaging of Ad DNA. These sequences include approximately 500 bp of viral DNA including the viral inverted terminal repeats (ITRs) and packaging signal ("psgr"), normally located at the left end of the genome. In the bacterial plasmid constructs, the left end of the left ITR is joined in a head-to-tail manner to the right ITR. Preferably, the helper-dependent viral vector also contains restriction enzyme sites suitable for the insertion of foreign DNA sequences. Optionally, the bacterial plasmids used to produce the helper-dependent vectors, may contain substantial deletions of the viral DNA sequences that are substituted with large insertions of foreign DNA, for a total size of up to 35 kb in length. Such genomes are unable to replicate as viruses in the absence of viral products provided by a helper virus.
It is another object of the invention to provide helper-dependent vectors having optimum DNA packaging lengths. In addition to possessing an upper packaging limit, the Ads of the present invention possess a lower packaging limit corresponding to approximately 75% of the wild-type genome length. Although DNAs with sizes less than this minimum are packaged, they apparently do so at a lower efficiency, resulting in a reduced virus recovery. Fisher et al. (1996) have developed a helper-dependent system utilizing a helper virus with a mutated packaging signal, and have used this system to amplify a vector of 5.5 kb. Analysis of the final viral stocks showed that the vector DNA had undergone DNA rearrangements and multimerization. This is consistent with our results showing that vectors less than xcx9c27 kb are packaged with a lower efficiency. Vector DNA which had undergone rearrangements, resulting in a net increase in size above this lower limit would be highly selected and would likely outgrow the smaller vector.
A third embodiment of the present invention is a mammalian cell line, such as a human cell line, that provides Cre recombinase. Alternatively, Cre may be provided by another source, such as a bacterial plasmid or Ad derived vector, that expresses the Cre protein in suitable cells.
A fourth embodiment of the invention, provides a mammalian cell line, such as a human cell line that expresses the Ad pIX protein. Alternatively, pIX may be provided by another source, such as a bacterial plasmid or Ad derived vector, that expresses the pIX protein in suitable cells.
In a preferred embodiment of the present invention, a helper virus is provided that contains a deletion or mutation of pIX coding sequences and has a genome of such a size that it cannot be packaged in the absence of pIX, but can be propagated under permissive conditions, and used to support replication of a second virus, i.e., the helper-dependent vector, from which substantial portions of the viral genome have been deleted and substituted with foreign DNA having an overall DNA size that can be packaged. Under nonpermissive conditions, i.e., in the absence of pIX, the helper virus DNA described herein is unable to be packaged into infectious virions but the helper-dependent vector DNA, being smaller than xcx9c35 kb in size, is able to be packaged into a virion capsid lacking pIX.
As previously described in copending patent application Ser. No. 08/473,168 now U.S. Pat. No. 5,919,676, packaging of the helper-dependent vector of the present invention can be enhanced relative to the helper virus DNA by the Cre-mediated removal of the viral packaging signal from the helper virus DNA. However, the invention described herein provides an improved helper-dependent vector system exhibiting reduced helper virus titers without the need for removal of the packaging signal from helper virus DNA as in the Cre/loxP system.
A fifth embodiment of the invention provides a kit for obtaining packaged helper-dependent vectors.